Post translational modifications (PTMs) of proteins are important in determining the epigenetic status of the genome. These modifications include phosphorylation, methylation, acetylation, glycosylation, ubiquitination, nitrosylation, and lipidation and influence many aspects of normal cell biology and pathogenesis. More specifically, histone related PTMs are of great importance as covalent modifications of histones have been implicated in transcriptional regulation via chromatin modulation. Examples of post translational modifying enzymes include, but are not limited to, Kinases/Phosphatases, Methyltransferases/Demethylases, Acetyltransferases/Histone Deacetylases, Glycosyltransferases/Glucanases and ADP-Ribosyl Transferases. Under normal physiological conditions, the regulation of PTM enzymes is tightly regulated. However, under pathological conditions, the activity of these enzymes can be dysregulated, and the disruption of the intracellular networks governed by these enzymes leads to many diseases including cancer and inflammation. Consequently, PTM enzymes have become an important target for drug discovery creating a need for technological development to monitor their activities. These assays can be applied not only to High-Throughput Screening (HTS) to search for drug candidates against these diseases, but also to understand the role of these post-translational modifications in regulating cellular processes.
JumonjiC domain-containing histone lysine demethylases (“JMJC demethylases”) play a role in determining the epigenetic status of the genome by counteracting the activities of histone lysine methyltransferases. These enzymes act as erasers by catalyzing the removal of methyl marks from specific lysine sites in histones, leading to either transcriptional repression or activation of target genes. JMJC demethylases are widely studied and, because of their implication in cancer, they have become validated drug targets. Thus, assays that monitor JMJC demethylase activities are desirable as basic research tools to elucidate their mode of regulation as well as to facilitate the identification of selective and potent inhibitors for drug discovery. Traditional JMJC demethylase assays use antibodies, enzyme-coupled assays, HPLC-based assays, or mass spectrometry to detect the substrates that have been demethylated by JMJC demethylases. These assays are not easily configured for rapid demethylase activity detection because they rely on the use of colorimetric, radioactive or fluorimetric non-homogenous antibody-based assays. Colorimetric assays are usually not desirable for drug discovery applications since they are prone to compound interference, low sensitivity, and higher rate of false hits. Radioactivity and mass spectrometry-based methods require sample processing. The use of radiolabeled materials requires waste management or cumbersome regulatory procedures. Because of the importance of these classes of enzymes, there is a need in developing enzymatic assays to monitor their activities, their regulation or identify novel inhibitors.